Ship inhibitors and uses thereof

ABSTRACT

The present invention relates to SHIP inhibitor compounds and methods for using these compounds. In particular, the present invention discloses the following methods: (i) a method of treating graft versus host disease in a subject; (ii) a method of inhibiting a SHIP1 protein in a cell; (iii) a method of selectively inhibiting a SHIP1 protein in a cell; (iv) a method for treating or preventing graft-versus-host disease (GVHD) in a recipient of an organ or tissue transplant; (v) a method of modulating SHIP activity in a cell expressing SHIP1 or SHIP2; (vi) a method of ex vivo or in vitro treatment of transplants; (vii) a method of inhibiting tumor growth and metastasis in a subject; (viii) a method of treating a hematologic malignancy in a subject; (ix) a method of inducing apoptosis of multiple myeloma cells; (x) a method of treating multiple myeloma in a subject; (xi) a method of inhibiting the proliferation of a human breast cancer cell; and (xii) a method of treating breast cancer in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. Provisional PatentApplication Ser. No. 61/322,378, filed Apr. 9, 2010, the disclosure ofwhich is hereby incorporated by reference herein in its entirety.

GOVERNMENT RIGHTS STATEMENT

This invention was made with Government support under grant numbersHL085580 and HL072523 awarded by the National Heart, Lung, and BloodInstitute of the National Institutes of Health. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to small molecule inhibitors of SHIP andtherapeutic uses of these inhibitors.

BACKGROUND OF THE INVENTION

Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP-1 orSHIP1) is a cytosolic protein that has been found to control theintracellular level of the phosphoinositide 3-kinase productphosphotidylinositol-3,4,5-trisphosphate and function as a negativeregulator of cytokine and immune receptor signaling. Using variousgenetic models, it has been shown that SHIP1 deficient hosts arepermissive for engraftment of major histocompatibility complex (MHC)mismatched bone marrow (BM) grafts, exhibit reduced GVHD post-transplantand delayed rejection of vascularized allogeneic heart grafts (Refs.1-5). In addition, SHIP1 deficiency profoundly increases myeloidimmunoregulatory (MIR) cell numbers and their function and granulocytenumbers (Refs. 2, 4, 6-8). These studies suggest that SHIP1 could betargeted to facilitate increase granulocyte/neutrophil numbers duringinfection or to reduce the severity and incidence of deleteriousallogeneic T cell responses in bone marrow and organ transplantation(Ref. 5).

SHIP1, SHIP2 and PTEN are commonly viewed as opposing the activity ofthe PI3K/Akt signaling axis that promotes survival of cancer cells andtumors. However, the enzymatic activities of these inositol phosphatasesare quite distinct in that the 3′-polyphosphatase activity of PTENreverses the PI3K reaction to generate PI(4,5)P₂ from PI(3,4,5)P₃, whilethe 5′-poly-phosphatase activity of SHIP1/2 converts PI(3,4,5)P₃ toPI(3,4)P₂. This distinction is potentially crucial as it might enableSHIP1/2 and PTEN to have distinctly different effects on Akt signaling.The PH domain of Akt binds with greater affinity to the SHIP1/2 productPI(3,4)P₂ leading to more potent activation of Akt than the directproduct of PI3K, PI(3,4,5)P₃ (Ref 9). Thus, SHIP1, which is expressed inmost blood cell malignancies, might actually contribute to their growthand survival. Consistent with this hypothesis, PI(3,4)P₂ levels areincreased in leukemia cells (Ref. 10) and increased levels of PI(3,4)P₂promote the transformation and tumorigenicity of mouse embryonicfibroblasts (MEF) (Ref. 11).

To date the molecular structure of SHIP1 has not been determined andthus a rational design approach to develop SHIP1 inhibitors has not beenfeasible. Thus, High-Throughput Screening (HTS) tests have been used toidentify compounds that can inhibit the enzymatic activity of SHIP1.However, there is a need for SHIP1 selective inhibitors that are capableof increasing granulocyte and MIR cell production in vivo and promotingapoptosis of blood cell cancers.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to SHIP inhibitor compoundsof the formula (I), and pharmaceutically acceptable salts thereof, whereformula (I) is as follows:

wherein the groups R¹, R², R³, R⁴, R⁵, R¹³, X¹, and X² are ashereinafter defined below.

The “SHIP inhibitor compounds” of the present invention are alsoreferred to herein as “SHIP inhibitors,” “SHIP1 inhibitors,” “SHIP1inhibitor compounds,” and the like. In one embodiment, the SHIPinhibitor compounds of the present invention are selective inhibitors ofSHIP 1.

R¹ is a straight chain C₁-C₄ alkyl or C₁-C₄ haloalkyl. In oneembodiment, R¹ is methyl.

R² is hydrogen, methyl, or halomethyl. In one embodiment, R² is methyl.

R³ is hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, C₁-C₄haloalkyl, or C₁-C₄ alkenyl. In one embodiment, both R³ and R¹³ arehydrogen.

R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, alkyl, orbenzyl. In one embodiment, R⁴ is hydrogen.

R⁵ includes a divalent oxo atom, or two hydrogen atoms, or one hydrogenatom together with an alkyl group. In one embodiment, R⁵ represents onehydrogen atom together with an alkyl group. In one embodiment, alkylgroup is 1,5-dimethylhexyl.

X¹ may be selected from the group consisting of hydrogen, hydroxy,mercapto, alkoxy, aryloxy, alkylthio, and arylthio. The alkoxy, aryloxy,alkylthio, and arylthio moieties may be further substituted.

X¹ may also be selected from the group consisting of alkylcarbonamido,arylcarbonamido, aminocarbonamido, hydrazinocarbonamido,alkylsulfonamido, arylsulfonamido, aminosulfonamido, andhydrazinosulfonamido, all of which may be further substituted.

X¹ may also be selected from the group consisting of (C₁-C₄alkyl)carbonyloxy, (C₁-C₄ alkoxy)carbonyloxy, arylcarbonyloxy,aryloxycarbonyloxy, and aminocarbonyloxy, all of which may be furthersubstituted.

X¹ may further be selected from the group consisting of a substituted orunsubstituted amino and secondary and tertiary amino groups that includeat least one C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclicsubstituent, or combinations thereof. In one embodiment, the secondaryor tertiary amino group contains at least one C₁-C₄ alkyl moiety, whichmay be further substituted.

X¹ may further be an aminoalkyl group, amino(CH₂)_(n), where “amino” isan unsubstituted or a substituted secondary or tertiary amino as definedabove, and n is an integer from 1 to 4.

X¹ may further represent a divalent oxygen moiety, ═O, or a divalentN-hydroxyamino moiety, ═NOH.

X¹ may further be an amino group, except when: R¹ and R² are eachmethyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents onehydrogen atom together with an alkyl group, where the alkyl group is1,5-dimethylhexyl alkyl group.

Each X² is independently defined to represent a divalent oxo or twohydrogen atoms. In one embodiment, each X² represents two hydrogenatoms.

In another aspect, the present invention relates to a method of treatinggraft versus host disease in a subject. This includes the step ofadministering a SHIP1 inhibitor of the present invention to a subject inneed of treatment.

In a further aspect, the present invention relates to a method ofinhibiting a SHIP1 protein in a cell. The method includes the step ofcontacting the cell containing a SHIP1 protein with a SHIP1 inhibitor ofthe present invention.

In yet another aspect, the present invention relates to a method ofselectively inhibiting a SHIP1 protein in a cell. The method includesthe step of contacting the cell containing a SHIP1 protein with a SHIP1inhibitor of the present invention, wherein the SHIP1 inhibitor of thepresent invention is provided in an amount effective to inhibit SHIP1but not to inhibit SHIP2 or PTEN.

In another aspect, the present invention relates to a method fortreating or preventing graft-versus-host disease (GVHD) in a recipientof an organ or tissue transplant. The method includes the step ofadministering to the transplant recipient a SHIP inhibitor of thepresent invention in a pharmaceutically effective amount after thetransplantation. In certain embodiments the step of administering theSHIP1 inhibitor is performed prior to the organ or tissue transplant.

In another aspect, the present invention relates to a method ofmodulating SHIP activity in a cell expressing SHIP1 or SHIP2. The methodincludes the step of contacting the cell with at least one SHIPinhibitor of the present invention. In a particular embodiment, the SHIPmodulation is used to prevent at least one disease selected from thegroup consisting of autoimmune disease, graft-versus-host disease, andsolid organ graft rejection, dietary-induced obesity, tumor cell growth.The modulated SHIP can be SHIP1 or SHIP2. In another embodiment, theSHIP modulation is used to prevent dietary-induced obesity. In furtherembodiments, the SHIP modulation can be used to modulate cell numbersand functions of cells selected from the group consisting ofhematopoietic stem cells, NK cells, Treg cells, and myeloid derivedsuppressor cells. The Treg cells are naive FoxP3+ T cells. In stillfurther embodiments, the SHIP modulation is used to convertnaive/effector CD4+ T cells into immunoregulatory cells. In furtherembodiments, the SHIP modulation can be used to facilitate engraftmentof cells selected from the group consisting of allogenic bone marrowstem cells, hematopoietic stem cells, pluripotent stem cells, IPS, andderivatives thereof.

In another aspect, the present invention relates to a method of ex vivoor in vitro treatment of transplants. The method can include the stepsof isolating blood derived cells, bone marrow transplants, or organtransplants and contacting the isolated blood derived cells, bone marrowtransplants, or organ transplants with a SHIP inhibitor of the presentinvention. The treatment of transplants serves to inactivateT-lymphocytes contained in the sample.

In another aspect, the present invention relates to a method ofinhibiting tumor growth and metastasis in a subject. The method includesthe step of administering to the subject one or more SHIP inhibitors ofthe present invention.

In another aspect, the present invention relates to a method of treatinga hematologic malignancy in a subject. The method includes the step ofadministering to the subject one or more SHIP inhibitors of the presentinvention. The hematologic malignancy can be a leukemia, lymphoma,multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferativedisease (MPD) or MDS/MPD diseases. In certain embodiments the leukemiais acute lymphoblastic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, or chronic lymphocytic leukemia. In certainembodiments the lymphoma is Hodgkin's disease, small lymphocyticlymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantlecell lymphoma, hairy cell leukemia, marginal zone lymphoma, Burkitt'slymphoma, Post-transplant lymphoproliferative disorder, T-cell prolymphocytic leukemia, B-cell prolymphocytic leukemia, Waldenstrom'smacroglobulinemiallymphoplasmacytic lymphoma, or other NK- or T-celllymphomas. In certain embodiments the myeloproliferative disease ispolycythemia vera, essential thrombocytosis or myelofibrosis. In certainembodiments the MDS/MPD disease is chronic myelomonocytic leukemia,juvenile myelomonocytic leukemia, and atypical chronic myeloid leukemia.

In another aspect, the present invention relates to a method of inducingapoptosis of multiple myeloma cells. The method includes the step ofcontacting the cells with one or more SHIP inhibitors of the presetinvention.

In another aspect, the present invention relates to a method of treatingmultiple myeloma in a subject. The method includes the step ofadministering to the subject one or more SHIP inhibitors of the presentinvention.

In another aspect, the present invention relates to a method ofinhibiting the proliferation of a human breast cancer cell. The methodincludes the step of contacting the cell with one or more SHIPinhibitors of the present invention.

In another aspect, the present invention relates to a method of treatingbreast cancer in a subject. The method includes the step ofadministering to the subject one or more SHIP inhibitors of the presentinvention.

In another aspect, the present invention relates to a method of treatingmyelosuppression in a subject. This method includes the step ofadministering to the subject one or more SHIP1 inhibitor compound of thepresent invention under conditions effective to treat myelosuppressionin the subject.

In another aspect, the present invention relates to a method of treatinganemia in a subject. This method includes the step of administering tothe subject one or more SHIP1 inhibitor compound of the presentinvention under conditions effective to treat anemia in the subject.

In another aspect, the present invention relates to a method ofincreasing platelets in a subject. This method includes the step ofadministering to the subject one or more SHIP1 inhibitor compound of thepresent invention under conditions effective to increase platelets inthe subject.

In another aspect, the present invention relates to a method of aidingrecovery of a subject who has undergone a bone marrow transplant. Thismethod includes the step of administering to the subject one or moreSHIP1 inhibitor compound of the present invention under conditionseffective to increase production in the subject of blood cellcomponents, thereby aiding the post-bone marrow transplant recovery ofthe subject. This method can be used to aid the recovery of subjects whohave undergone an autologous bone marrow transplant or an allogenic bonemarrow transplant. In one embodiment, this method can further includeadministering at least one growth factor to the subject along with theone or more SHIP1 inhibitor compound.

In another aspect, the present invention relates to a method ofenhancing blood stem cell harvest from a subject. This method includesthe step of administering to the subject one or more SHIP1 inhibitorcompound of the present invention under conditions effective to mobilizestem cells in the subject from the bone marrow to the blood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a SHIP1 malachite phosphatase assay. Lack ofgreen color indicates inhibition for the amide and the hydrochloride,while the ethylamine is a poor inhibitor

FIG. 2 shows the results of a SHIP1 malachite phosphatase assay. Lack ofgreen color indicates inhibition.

FIG. 3 shows the results of a SHIP1 malachite phosphatase assay using aUV-Vis plate reader. Absorbance is recorded at 620 nm, smaller numbersindicate a lack of green color and inhibition of the phosphatase.

FIG. 4 shows the results of a SHIP1 malachite phosphatase assay using aUV-Vis plate reader. Absorbance is recorded at 620 nm, smaller numbersindicate a lack of green color and inhibition of the phosphatase.

FIGS. 5A-5C illustrate results of potency studies regarding3α-amino-5α-androstane (3A5AS). The 3AC derivative, 3A5AS (FIG. 5A), isequally potent for inhibition of recombinant SHIP1 activity in vitro asmeasured by a Malachite Green assay (see FIG. 5B). However, 3A5AS ismore potent for killing of blood cancer cells (C1498 leukemia cells)than the parent compound 3AC as measured in the MTT assay for cellviability (see FIG. 5C).

FIGS. 6A-6D illustrate results relating to SHIP1 inhibitory activity ofa 3AC derivative. As shown by the results, a more soluble 3AC derivativeretains SHIP1 inhibitory activity in vitro and in vivo. FIG. 6A:Malachite Green assay for SHIP1 activity against PI(3,4,5)P₃ substratein the presence of 3AC or 3A5AS. The “No SHIP” column indicatesbackground absorbance when the assay is carried out with PIP3 substratein the absence of recombinant SHIP1. FIG. 6B: MTT assay of C1498leukemia growth in the presence of 3AC and 3A5AS at the indicatedconcentrations. FIG. 6C: Examples of MIR cell induction in 3AC- and3A5AS-treated mice as compared to a vehicle control. FIG. 6D: Bar graphsand statistical analysis of MIR cell numbers in the indicated treatmentgroups (**, p<0.01; NS, not significant). [Noto bene: DRV-IV-26=3A5AS]

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to SHIP inhibitor compounds of the formula(I), and pharmaceutically acceptable salts thereof, where formula (I) isas follows:

wherein the groups R¹, R², R³, R⁴, R⁵, R¹³, X¹, and X² are ashereinafter defined.

The “SHIP inhibitor compounds” of the present invention are alsoreferred to herein as “SHIP inhibitors,” “SHIP1 inhibitors,” “SHIP1inhibitor compounds,” and the like.

In one embodiment, the SHIP inhibitor compounds of the present inventionare selective inhibitors of SHIP1.

R¹ is a straight chain C₁-C₄ alkyl or C₁-C₄ haloalkyl. In oneembodiment, R¹ is methyl.

R² is hydrogen, methyl, or halomethyl. In one embodiment, R² is methyl.

R³ is hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, C₁-C₄haloalkyl, or C₁-C₄ alkenyl. In one embodiment, both R³ and R¹³ arehydrogen.

R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, alkyl, orbenzyl. In one embodiment, R⁴ is hydrogen.

R⁵ includes a divalent oxo atom, or two hydrogen atoms, or one hydrogenatom together with an alkyl group. In one embodiment, R⁵ represents onehydrogen atom together with an alkyl group. In one embodiment, alkylgroup is 1,5-dimethylhexyl.

X¹ may be selected from the group consisting of hydrogen, hydroxy,mercapto, alkoxy, aryloxy, alkylthio, and arylthio. The alkoxy, aryloxy,alkylthio, and arylthio moieties may be further substituted.

X¹ may also be selected from the group consisting of alkylcarbonamido,arylcarbonamido, aminocarbonamido, hydrazinocarbonamido,alkylsulfonamido, arylsulfonamido, aminosulfonamido, andhydrazinosulfonamido, all of which may be further substituted.

X¹ may also be selected from the group consisting of (C₁-C₄alkyl)carbonyloxy, (C₁-C₄ alkoxy)carbonyloxy, arylcarbonyloxy,aryloxycarbonyloxy, and aminocarbonyloxy, all of which may be furthersubstituted.

X¹ may further be selected from the group consisting of a substituted orunsubstituted amino and secondary and tertiary amino groups that includeat least one C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclicsubstituent, or combinations thereof. In one embodiment, the secondaryor tertiary amino group contains at least one C₁-C₄ alkyl moiety, whichmay be further substituted.

X¹ may further be an aminoalkyl group, amino(CH₂)_(n), where “amino” isan unsubstituted or a substituted secondary or tertiary amino as definedabove, and n is an integer from 1 to 4.

X¹ may further represent a divalent oxygen moiety, ═O, or a divalentN-hydroxyamino moiety, ═NOH.

X¹ may further be an amino group, except when: R¹ and R² are eachmethyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents onehydrogen atom together with an alkyl group, where the alkyl group is1,5-dimethylhexyl alkyl group.

Each X² is independently defined to represent a divalent oxo or twohydrogen atoms. In one embodiment, each X² represents two hydrogenatoms.

The compounds of the present invention, as will be appreciated by oneskilled in the art, possess several potential chiral carbon atoms. As aconsequence of these chiral centers, the compounds of the presentinvention may occur as racemates, racemic mixtures, individualdiastereomers and substantially pure isomers. All asymmetric forms,individual isomers, and combinations thereof, are within the scope ofthe present invention.

Throughout this specification, the terms and substituents retain theirdefinitions. Below are particular definitions of terms used herein.

The term “alkyl” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain hydrocarbonradical having the stated number of carbon atoms and includes straightor branch chain groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, and higher homologs and isomerssuch as n-pentyl, n-hexyl, 2-methylpentyl, 1,5-dimethylhexyl,1-methyl-4-isopropyl, hexyl and the like. A divalent radical derivedfrom an alkane is exemplified by —CH₂CH₂CH₂CH₂—. A divalent radicalderived from an alkene is exemplified by —CH═CH—CH₂—.

The term “alkenyl”, employed alone or in combination with other terms,means a straight chain or branched monounsaturated hydrocarbon grouphaving the stated number of carbon atoms, such as, for example, vinyl,propenyl (allyl), crotyl, isopentenyl, and the various butenyl isomers.

Alkyl and alkenyl groups may include substitutents selected from thegroup consisting of halo, hydroxy, cyano, mercapto, —S(C₁-C₄ alkyl),amino, substituted amino, acetamido, carboxy, trifluoromethyl, C₁-C₄alkoxy, (C₁-C₄ alkoxy)carbonyl and aminocarbonyl.

The term “cycloalkyl” means an unsubstituted or substituted monovalentsaturated cyclic hydrocarbon radical having the stated number of carbonatoms, including, various isomers of cyclopentyl and cyclohexyl. Theterm “cycloalkenyl” means an unsubstituted or substituted monovalentmonounsaturated cyclic hydrocarbon radical having the stated number ofcarbon atoms, including, various isomers of cyclopentenyl andcyclohexenyl. The term “cycloalkadienyl” means a monovalentdiunsaturated cyclic radical having the stated number of carbon atoms,including, the various isomers of cyclopentadienyl and cyclohexadienyl.The substituents can be one or two of the same or different substituentsselected from halo, hydroxy, cyano, mercapto, —S(C₁-C₄ alkyl), amino,substituted amino, acetamido, carboxy, trifluoromethyl, C₁-C₄ alkoxy,(C₁-C₄ alkoxy)carbonyl and aminocarbonyl.

The dotted lines between the 4,5 and 5,6 positions represent thepresence or absence of an additional bond; that is, an unsaturation.Only one unsaturation can be present at any one time. The 5 positionhydrogen atom and R¹³ shown in Formula (I) will, of course, be absentwhen an unsaturation is present.

The term “aryl” means an unsubstituted or substituted monovalent phenylgroup. The substituents may be independently selected from halo, —OH,—SH, —S(C₁-C₄) alkyl), C₁-C₅ alkyl, C₁-C₅ alkoxy, carboxy, (C₁-C₄alkoxy)carbonyl, aminocarbonyl, C₁-C₄ alkylaminocarbonyl, amino,acetamido, C₁-C₄ alkylamino, di(C₁-C₄ alkyl)amino or a group—(CH₂)_(q)—R where q is 1, 2, 3, or 4 and R is hydroxy, C₁-C₄ alkoxy,carboxy, C₁-C₄ alkoxycarbonyl, amino, aminocarbonyl, C₁-C₄ alkylamino ordi(C₁-C₄ alkyl)amino.

The term “benzyl” means a monovalent group in which a phenyl moiety issubstituted by a methylene group. The benzyl group may include furthersubstituents on the phenyl moiety.

The term “amino” means a group —NH₂. The term, “substituted amino” meansan amino group where one or both amino hydrogens are independentlyreplaced by a C₁-C₄ alkyl, C₂-C₄ alkenyl, C₅-C₆ cycloalkyl, C₅-C₆cycloalkenyl, aryl, benzyl, or a group —(CH₂)_(q)—R where q is 1, 2, 3,or 4 and R is hydroxy, C₁-C₄ alkoxy, carboxy, C₁-C₄ alkoxycarbonyl,amino, aminocarbonyl, C₁-C₄ alkylamino or di(C₁-C₄ alkyl)amino.

The term “alkylcarbonamido” means a group (C₁-C₄ alkyl)C(O)N(R)—, whereR represents H or C₁-C₄ alkyl. More specifically, the term “acetamido”means a group CH₃C(O)NH—. The term “arylcarbonamido” means a group(aryl)C(O)N(R)—, where R represents H or C₁-C₄ alkyl. The term“aminocarbonamido” means a group R′R″NC(O)N(R)—, where R represents H orC₁-C₄ alkyl, and R′ and R″ independently represent H, C₁-C₄ alkyl, C₅-C₆cycloalkyl, aryl, or heterocyclic.

The term “alkylsulfonamido” means a group (C₁-C₄ alkyl)SO₂N(R)—, where Rrepresents H or C₁-C₄ alkyl. The term “arylsulfonamido” means a group(aryl)SO₂N(R)—, where R represents H or C₁-C₄ alkyl. The term“aminosulfonamido” means a group R′R″NHSO₂N(R)—, where R represents H orC₁-C₄ alkyl, and R′ and R″ independently represent H, C₁-C₄ alkyl, C₅-C₆cycloalkyl, aryl, or heterocyclic.

The term “alkylcarbonyloxy” means a group (C₁-C₄ alkyl)C(O)O—. The term“alkoxycarbonyloxy” means a group (C₁-C₄ alkyl)OC(O)O—. The term“arylcarbonyloxy” means a group (aryl)C(O)O—. The term“aryloxycarbonyloxy” means a group (aryl)OC(O)O—. The term“aminocarbonyloxy” means a group R′R″NC(O)O—, where R′ and R″independently represent H, C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, orheterocyclic.

The term “halo” means chloro, bromo, fluoro or iodo. The term “mercapto”means a group —SH.

The term “heterocycle” means an unsubstituted or substituted stable 5-or 6-membered monocyclic heterocyclic ring that consists of carbon atomsand from one to three heteroatoms selected from the group consisting ofN, O and S, and wherein the nitrogen and sulfur heteroatoms mayoptionally be oxidized, and the nitrogen heteroatom may optionally bequaternized. The heterocyclic ring may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. The heterocycle may be unsubstituted or substituted with oneor two substituents.

In one embodiment of the present invention, the compound of formula (I)is a compound of a formula as set forth below:

and pharmaceutically acceptable salts thereof, wherein X═NR2, NRCOR,NHCONR2, OR, SR, OCOR, OCONR2, or NHCNHNH2, and wherein R═H, alkyl,cycloalkyl, aryl, or benzyl.

The present invention provides methods for prevention and clinicaltreatment of various forms of graft-versus-host disease (GVHD) by usinginhibitors of SH2-domain containing inositol phosphatase (SHIP). Inparticular, novel formulations of SHIP inhibitors are provided for thetreatment in order to suppress T-lymphocyte mediated immune responses.

SHIP1-deficiency has been linked to transplant tolerance in geneticstudies. Accordingly, molecular targeting of SHIP1 can be utilized toachieve similar effects, including an increase in immunoregulatorycapacity. The SHIP1 inhibitor compounds of the present invention providesuch molecular targeting. Treatment with the SHIP1 inhibitor compoundsof the present invention significantly expands the myeloidimmunoregulatory cell compartment and impairs the ability of peripherallymphoid tissues to prime allogeneic T cell responses. In addition,treatment with the SHIP1 inhibitor compounds of the present inventionprofoundly increases granulocyte production without triggering themyeloid-associated lung consolidation observed in SHIP1^(+/−) mice. Itwas also found that chemical inhibition of SHIP1 triggers apoptosis ofblood cancer cells. Thus, SHIP1 inhibitors of the present inventionprovide a novel class of small molecules that have the potential toenhance allogeneic transplantation, boost innate immunity, and improvethe treatment of hematologic malignancies.

SHIP is critical in cell-mediated allogeneic immune responses, and SHIPdeficient hosts do not support priming of allogeneic T cell responses.Targeting SHIP facilitates allogeneic transplantation. It is alsobelieved that the SHIP1 inhibitor compounds of the present invention canalso inhibit SHIP2, a potential molecular target in diabetes, but notPTEN. It is further believed that the SHIP1 inhibitor compounds of thepresent invention can also inhibit the ability of peripheral lymphoidtissues to prime allogeneic T cell responses in vitro. It is believedthat administration of the SHIP1 inhibitor compounds of the presentinvention expands the number of both myeloid and T lymphoidimmunoregulatory cells in secondary lymphoid tissues where GvHD isprimed and expands the number of NK cells in the periphery of models.Such results demonstrate the enzymatic activity of SHIP is required forthe priming of allogeneic T cells responses.

The SHIP1 inhibitor compounds of the present invention described andidentified herein are believed to significantly inhibit the enzymaticactivity of SHIP in solution. To further validate that compoundsidentified in the solution based assay for SHIP activity are cellpermeable and can alter the immune system in a manner comparable to thatobserved in SHIP deficient mice, the ability of some of the more potentSHIP inhibitors can be tested to inhibit priming of an allogeneic T cellresponse in vitro and for the ability to expand immunoregulatory cellpopulations and to abrogate GvHD. A potent inhibitor of SHIP in solutionis also shown to inhibit priming of an allogeneic T cell response asmeasured in an MHC-mismatched MLR and can significantly expand thenumber of myeloid and T lymphoid immunoregulatory cells in secondarylymphoid tissues.

That SHIP inhibitors identified via a HTS screen can impair priming ofallogenic T cells responses in vitro and can expand immunoregulatorycells in lymphoid tissues suggests that chemical inhibition of SHIPactivity could be utilized to facilitate allogeneic transplantationprocedures. Therefore, the SHIP1 inhibitor compounds of the presentinvention are useful to enhance engraftment of allogeneic BM as Tregcells are known to not only combat GvHD, but can also facilitateengraftment of donor BM in MHC-mismatched transplant settings. Inaddition, expansion of myeloid derived suppressor cell (MDSC) and Tregcell numbers also reduces the frequency that donor T cells are primed byhost antigen presenting cells (APC) in secondary lymphoid tissues and,thus reduces the incidence and severity of GvHD. As solid organ graftresponses by host T cells are also primed in secondary lymphoid tissues,and Treg cells also facilitate solid organ graft acceptance, the SHIPinhibitors of the present invention will prove useful for reducing organgraft rejection. As SHIP-deficient mice exhibit normal humoral immunityand APC priming of T cell response to foreign antigens, the compoundsdescribed herein spare normal adaptive immune function. Thus, they offera more selective method to dampen deleterious host and donor allogenic Tcell responses without compromising adaptive immune functions necessaryto combat opportunistic pathogens that frequently infect transplantpatients undergoing conventional immunosuppressive therapies.

SHIP inhibition also prevents chemo-attraction of tumor cells todirected tissues in vivo. SDF1 serves as a chemo-attractant to lure stemcells and tumor cells into tissue sites, referred to as metastasis fortumor cells. There is very little or no SDF1/CXCL 12 produced in BM(bone marrow) or solid organs (e.g. spleen) in SHIP-deficient mice.Thus, SHIP inhibitors may be administered to shut down or significantlyreduce production of SDF1/CXCL 12 in tissues and organs. The SHIPinhibitors are also useful to inhibit tumor growth and metastasis insolid organs and tissues.

The present disclosure further describes the identification and initialin vivo characterization of small molecule inhibitors of the SHIP1enzyme. To validate that these compounds are cell permeable and canalter the immune system in a manner comparable to that observed in SHIP1deficient mice, their ability to expand MIR cells and to consequentlyinhibit priming of an allogeneic T cell response was tested. It is shownherein that chemical inhibition of SHIP1 is capable of both. Inaddition, SHIP1 inhibition promotes a profound increase in circulatinggranulocyte numbers and apoptosis of blood cancer cells.

It is also shown that administration of a SHIP1 inhibitor can expandimmunoregulatory cells in peripheral lymphoid tissues and suppresspriming of allogeneic T cell responses. Because allogeneic T cellresponses that culminate in GvHD or solid organ graft rejection areprimed in peripheral lymphoid tissues, [Lafferty K J, et al., Surg ClinNorth Am (1986) 66(6):1231-1253; Kosaka H, et al., J Exp Med (1992)176(5):1291-1302; Shlomchik W D, et al., Science (1999)285(5426):412-415] these results show that the SHIP inhibitor compoundsof the present invention can potentially be used to limit deleterious Tcell responses that mediate GvHD and organ graft rejection. Consistentwith this, GvHD is reduced and cardiac graft rejection delayed in adultmice rendered SHIP1-deficient [Paraiso K H, et al., J Immunol (2007)178(5):2893-2900; Collazo M M, et al., Blood (2009) 113:2934-2944]. AsSHIP1-deficient mice exhibit normal humoral immunity [Brauweiler A, etal., J Exp Mad (2000) 191(9):1545-1554; Liu Q, et al., J Exp Med (1998)188(7):1333-1342] and priming of T cell responses to naive antigens[Ghansah T, et al., J Immunol (2004) 173(12):7324-7330], the SHIP1inhibitor described here, and potentially others, may not significantlycompromise adaptive immune function. Thus, the SHIP1 inhibitor compoundsof the present invention offers a more selective method to dampendeleterious host and donor allogeneic T cell responses withoutcompromising adaptive immune functions necessary to combat opportunisticpathogens that can compromise the recovery and survival of transplantpatients receiving state-of-the-art immunosuppressive regimens.

Increased Akt signaling and survival in primary NK [Wang J W, et al.,Science (2002) 295(5562):2094-2097] and myeloid cells [Liu A, et al.,Genes & Development (1999) 13(7):786-791] isolated from SHIP1^(+/−) micehave been documented. However, there is also an emerging role for theSHIP1/2 product PI(3,4)P₂ in promoting Akt activation [Franke T F, etal., Science (1997) 275(5300):665-668] and tumorigenicity. [Ivetac I.,et al., EMBO Rep (2009) 10(5):487-493] Thus, via generation ofPI(3,4)P₂, SHIP1/2 could amplify survival signals in transformed orneoplastic cells by providing additional plasma membrane locations forrecruitment and activation of PH-domain containing kinases, such as Akt.Indeed, PI(3,4)P₂ levels are found to be increased in leukemia cells.[Jain S K, et al., Blood (1996) 88(5):1542-1550] Consistent with thishypothesis, it is shown that a SHIP1 selective inhibitor reduces Aktactivation and promotes apoptosis of human blood cell cancers thatexpress SHIP1. Thus, SHIP1 inhibition can be used as an adjunct to othertherapeutics to further decrease the survival of hematologicmalignancies. There will also be applications for SHIP1/2 inhibitors innon-hematologic cancers as SHIP2 expression is increased in breastcancer and promotes survival signals from EGF-R in these cells. [PrasadN K, et al., Tumour Bioi (2008) 29(5):330341; Prasad N K, et al.,Carcinogenesis (2008) 29(1):25-34; Prasad N K, Int J Onool (2009)34(1):97-105]

Although treatment of mice with a SHIP1 selective inhibitor induced manyof the same myeloid phenotypes observed in mice that are geneticallySHIP1-deficient, some key deleterious effects associated with geneticSHIP1 deficiencies were notably absent. Importantly, we did not toobserve myeloid lung consolidation and pneumonia emerging ininhibitor-treated mice. This could be fortuitous, since this pneumoniais the major pathology that limits the lifespan of SHIP1^(+/−) mice.[Helgason C D, et al. (1998) Genes & Development 12(11):1610-1620]Without wishing to be bound to a particular theory, there are severalreasons that chemical inhibition of SHIP1 enzymatic activity andgermline SHIP1 deficiency do not result in identical hematologicmanifestations. In germline SHIP1-deficient mice there is complete lossof SHIP1 protein from the point of conception and, thus, thedevelopmental effects of SHIP1-deficiency may trigger some abnormalitiesthat may not occur in the treatment of adult mice with a SHIP1inhibitor. Although it has been documented that several SHIP1 phenotypesare induced in MxCreSHIP1^(flox/flox) mice rendered SHIP1-deficient asadults, [Ghansah T, et al., J Immunol (2004) 173(12):7324-7330; Hazen AL, et al., Blood (2009) 113(13):2924-2933; Collazo M M, et al., Blood(2009) 113:2934-2944] these mice have not been examined for lungpathology. Another possible explanation for the difference betweenchemical and genetic ablation of SHIP1 function is that a SHIP1 nullmutation results in the absence of SHIP1 protein. The absence of SHIP1protein has the potential to permit inappropriate activities by othersignaling proteins that assume its place in cell signaling complexes. Infact, this is known to occur in SHIP1^(+/−) NK cells, as loss of SHIP1expression leads to inappropriate recruitment of SHP1 to the 2B4 SLAMfamily receptor converting this receptor from activating mode to adominant inhibitory mode. [Wahle J A, et al., J Immunol (2007)179(12):8009-8015] It is possible then that the myeloid lungconsolidation observed in SHIP1^(+/−) mice also results frominappropriate activity by another signaling protein that fills the voidleft by the absence of SHIP1 protein. Further analysis of thesequestions could provide mechanistic insights into the role that SHIP1plays in alveolar macrophage biology.

In addition to the above effects relevant to allogeneic transplantation,SHIP1 inhibitors will also offer benefits to cancer patients. Forinstance, a SHIP1 inhibitor could be used to enhance granulocyterecovery after autologous BMT or high dose chemo/radiotherapy thatfrequently compromises granulocyte production and function. Granulocytesserve as the first line of defense against bacterial, fungal andparasitic infections and thus play a prominent role in recoveryfollowing myeloablative therapies. In addition, the growth and survivalof SHIP1-expressing blood cell malignancies is significantly reduced bychemical inhibition of SHIP1. Thus, the SHIP1 inhibitor compounds of thepresent invention represent a novel class of compounds that couldpotentially find utility in both transplantation and the treatment ofcancer.

In one embodiment, a method is provided for treating a patient sufferingfrom GVHD. The method comprises administering to the GVHD patient acomposition including a SHIP1 inhibitor compound of the presentinvention.

Dosage amounts and frequency will vary according to the particular SHIPinhibitor, the dosage form, and individual patient characteristics.Generally speaking, determining the dosage amount and frequency for aparticular SHIP inhibitor, dosage form, and individual patientcharacteristic can be accomplished using conventional dosing studies,coupled with appropriate diagnostics.

In a particular embodiment, a SHIP inhibitor is used to treat patientsthat have acute Graft vs Host Disease (aGVHD) but failed at least oneimmunosuppressive regimen such as a regimen including steroids such asprednisone and methylprednisolone, cyclophosphamide, cyclosporin A,FK506, thalidomide, azathioprine, and daclizumab. For example,hematopoietic stem cell transplant (HSCT) patients manifesting grade 2or greater aGVHD, who have failed to respond to treatment with at least2 mg/Kg of methylprednisolone or equivalent corticosteroid or othersalvage therapy, can be treated with a SHIP1 inhibitor.

A SHIP inhibitor of the present invention can also be used as aprophylaxis to prevent onset of GVHD or to reduce the effects of GVHD.

A SHIP inhibitor of the present invention may be administered as a GVHDprophylaxis to a transplant recipient within a predetermined time windowbefore or after the transplantation.

In one embodiment, a SHIP inhibitor of the present invention may beadministered to the recipient on days −3 or −2 (i.e., 3 or 2 days beforethe transplantation) as part of a non-myeloablative conditioningregimen, then followed by transplantation such as hematopoietic stemcell infusion. Alternatively, a SHIP inhibitor of the present inventionmay be administered as a GVHD prophylaxis to a transplant recipientafter the transplantation. For example, for standard (i.e.,myeloablative) transplant or non-myeloablative stem cell transplant(NST) where a SHIP inhibitor of the present invention is not used in theconditioning regimen, a SHIP inhibitor of the present invention isadministered to the transplant recipient at 0.5-1.5 mg/m²/day on days+8, +15, +22, and +30 following stem cell infusion.

Besides use in a single-agent treatment or prevention of GVHD, a SHIPinhibitor of the present invention can also be used in a combinationtherapy for acute or chronic GVHD. The combination therapy may havesynergistic therapeutic effects on the patients and thus requires loweramount of the SHIP inhibitor of the present invention and the otheragent used in conjunction to achieve satisfactory therapeutic efficacy.As a result, potential side effects associated with high dose of drugs,such as myelosuppression, are reduced and the patient's quality of lifeis improved.

Various other therapeutic agents may be combined with the SHIP inhibitorfor the treatment or prevention of GVHD. The other therapeutic agentsinclude, but are not limited to, immunosuppressive agents such assteroids (e.g., prednisone and methylprednisolone), cyclophosphamide,cyclosporin A, FK506, thalidomide, azathioprine, monoclonal antibodies(e.g., Daclizumab (anti-interleukin (IL)-2), Infliximab (anti-tumornecrosis factor), MEDI-205 (anti-CD2), abx-cbl (anti-CD147), andpolyclonal antibodies (e.g., ATG (anti-thymocyte globulin)). Forexample, a SHIP inhibitor of the present invention may be combined witha steroid such as methylprednisolone to treat GVHD. However, such acombination may be too broadly immunosuppressive to render the patientmore susceptible to opportunistic infection.

For the treatment of acute GVHD, a SHIP inhibitor of the presentinvention may preferably be combined with monoclonal antibodies whichspecifically target T-cells such as Infliximab, Daclizumab, MEDI-205, orabx-cbl. The monoclonal antibody may be administered at the FDA-approveddosage and by its standard route of administration (e.g., IV), followedby oral or parenteral administration of a SHIP inhibitor of the presentinvention.

The SHIP inhibitor may also be used in conjunction with otherimmunosuppressive agents as prophylaxis for GVHD post-transplantation.For example, the recipient of bone marrow transplant may be treated witha SHIP inhibitor of the present invention in conjunction with a standardpost infusion regimen including mini-methotrexate at 5 mg/m² (as opposedto the conventional dose at 10-15 mg/m²), cyclosporine A (5-6 mg/Kg/d IVor 10-18 mg/Kgld orally) and FK506 (0.05-0.1 mglKg/d IV or 0.15-0.3mg/Kg/d orally).

In addition, a SHIP inhibitor of the present invention may be used inconjunction with other types of therapy as prophylaxis for GVHD prior totransplantation. For example, the recipient of bone marrow transplantmay be pretreated with a SHIP inhibitor of the present invention inconjunction with TBI (radiation), phototherapy, melphalan,cyclophosphamide or ATG to prevent the onset of GVHD.

In yet another aspect, the invention relates to a method of ex vivo orin vitro treatment of blood derived cells, bone marrow transplants, orother organ transplants. The method comprises treating the blood derivedcells, bone marrow transplants, or other organ transplants with a SHIPinhibitor of the present invention in an effective amount such thatactivities of T-lymphocytes therein are substantially inhibited,particularly by at least 50% reduction in activity, more particularly byat least 80% reduction in activity, and further more particularly by atleast 90% reduction in activity.

The invention is practiced in an in vitro or ex vivo environment. All ofthe discussion above regarding clinical treatment or prevention of GVHDthat is relevant to an in vitro or ex vivo environment applies to thispractice. In a particular embodiment, practice of an in vitro or ex vivoembodiment of the invention might be useful in the practice of immunesystem transplants, such as bone marrow transplants or peripheral stemcell procurement. In such procedures, the SHIP inhibitor might be used,as generally described above, to treat the transplant material toinactivate T-lymphocytes therein so that the T-lymphocyte mediatedimmune response is suppressed upon transplantation.

For example, a SHIP inhibitor of the present invention may be added to apreservation solution for an organ transplant in an amount sufficient toinhibit activity of T-lymphocytes of the organ. Such a preservationsolution may be suitable for preservation of different kind of organssuch as heart, kidney and liver as well as tissue therefrom. An exampleof commercially available preservation solutions is Plegisol (Abbott),and other preservation solutions named in respect of its origins includethe UW-solution (University of Wisconsin), the Stanford solution and theModified Collins solution. The preservation solution may also containconventional co-solvents, excipients, stabilizing agents and/orbuffering agents.

The dosage form of the SHIP inhibitor of the present invention may be aliquid solution ready for use or intended for dilution with apreservation solution. Alternatively, the dosage form may be lyophilizedor power filled prior to reconstitution with a preservation solution.The lyophilized substance may contain, if suitable, conventionalexcipients.

The preservation solution or buffer containing a SHIP inhibitor of thepresent invention may also be used to wash or rinse an organ transplantprior to transplantation or storage. For example, a preservationsolution containing pentostatin may be used to flush perfuse an isolatedheart which is then stored at 4°.

In another embodiment, practice of the invention might be used tocondition organ transplants prior to transplantation. Prior totransplantation a SHIP inhibitor of the present invention may be addedto the washing buffer to rid the transplant of active T-lymphocytes. Inthis way, the risk of developing acute GVHD upon transplantation shouldbe significantly reduced, and the host is not only protected from GVHDbut also from potential side effects of the SHIP inhibitor. Theconcentration of the SHIP inhibitor in the preservation solution or washbuffer may vary according to the type of transplant. Other applicationsin vitro or ex vivo using a SHIP inhibitor of the present invention willoccur to one of skill in the art and are therefore contemplated as beingwithin the scope of the invention.

Other than in the operating examples, or unless otherwise expresslyspecified, all of the numerical ranges, amounts, values and percentagessuch as those for amounts of materials, times and temperatures ofreaction, ratios of amounts, values for molecular weight (whether numberaverage molecular weight (“M_(n)”) or weight average molecular weight(“M_(w)”), and others in the following portion of the specification maybe read as if prefaced by the word “about” even though the term “about”may not expressly appear with the value, amount or range. Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Furthermore, when numerical ranges ofvarying scope are set forth herein, it is contemplated that anycombination of these values inclusive of the recited values may be used.

As used herein, the term “pretreating” (or “pretreatment”) is intendedto mean that a first treatment is administered prior to, or inconjunction with, a second treatment. In other words, the pretreatmentmay be performed before another, later treatment, thus allowing thepretreatment time to take effect. Alternatively, the pretreatment may beperformed or administered simultaneously with a second treatment withouta temporal delay. Advantageously, a pretreatment is administered priorto a second treatment.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prod rug thereof and other agents.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician. In reference to cancers or other unwanted cellproliferation, an effective amount comprises an amount sufficient tocause a tumor to shrink and/or to decrease the growth rate of the tumor(such as to suppress tumor growth) or to prevent or delay other unwantedcell proliferation. In some embodiments, an effective amount is anamount sufficient to delay development. In some embodiments, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore doses. In the case of cancer, the effective amount of the drug orcomposition may: (i) reduce the number of cancer cells; (ii) reducetumor size; (iii) inhibit, retard, slow to some extent and preferablystop cancer cell infiltration into peripheral organs; (iv) inhibit(i.e., slow to some extent and preferably stop) tumor metastasis; (v)inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrenceof tumor; and/or (vii) relieve to some extent one or more of thesymptoms associated with the cancer.

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

As used herein, “treatment” refers to obtaining beneficial or desiredclinical results. Beneficial or desired clinical results include, butare not limited to, anyone or more of: alleviation of one or moresymptoms (such as tumor growth or metastasis), diminishment of extent ofcancer, stabilized (i.e., not worsening) state of cancer, preventing ordelaying spread (e.g., metastasis) of the cancer, preventing or delayingoccurrence or recurrence of cancer, delay or slowing of cancerprogression, amelioration of the cancer state, and remission (whetherpartial or total). The methods of the invention contemplate anyone ormore of these aspects of treatment.

A “subject in need of treatment” is a mammal with a condition that islife-threatening or that impairs health or shortens the lifespan of themammal.

A “pharmaceutically acceptable” component is one that is suitable foruse with humans and/or animals without undue adverse side effects (suchas toxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio.

A “safe and effective amount” refers to the quantity of a component thatis sufficient to yield a desired therapeutic response without undueadverse side effects (such as toxicity, irritation, or allergicresponse) commensurate with a reasonable benefit/risk ratio when used inthe manner of this invention.

A “pharmaceutically acceptable carrier” is a carrier, such as a solvent,suspending agent or vehicle, for delivering the compound or compounds inquestion to the animal or human. The carrier may be liquid or solid andis selected with the planned manner of administration in mind. Liposomesare also a pharmaceutical carrier. As used herein, “carrier” includesany and all solvents, dispersion media, vehicles, coatings, diluents,antibacterial and antifungal agents, isotonic and absorption delayingagents, buffers, carrier solutions, suspensions, colloids, and the like.The use of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions is contemplated.

A person of ordinary skill in the art can easily determine anappropriate dose of one of the instant compositions to administer to asubject without undue experimentation. Typically, a physician willdetermine the actual dosage which will be most suitable for anindividual patient and it will depend on a variety of factors includingthe activity of the specific compound employed, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, the severity of the particular condition, and theindividual undergoing therapy. The dosages disclosed herein areexemplary of the average case. There can of course be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods. See,generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed,John Wiley & Sons, Inc.; as well as Guthrie et al., Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Vol. 194,Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods andApplications (Innis, et al. 1990. Academic Press, San Diego, Calit),McPherson et al., PCR Volume 1, Oxford University Press, (1991), Cultureof Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney.1987. Liss, Inc. New York, N.Y.), and Gene Transfer and ExpressionProtocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc.,Clifton, N.J.).

In addition to the applications described above, the SHIP inhibitorcompounds of the present invention can be used for various otherapplications, particularly regarding treatment of conditions that followchemotherapy, radiation therapy, or infection, or that occur inmyelodysplastic/bone failure patients. Such conditions can include,without limitation, myelosuppression, anemia, and the lack of platelets.For example, the SHIP inhibitors of the present invention may be used toboost production of all key blood cell types as well as white bloodcells and lymphocytes. The SHIP inhibitors of the present invention mayalso be used for patients recovering from either an autologous orallogenic bone marrow transplant to enhance recovery of key blood cellcomponents after transplant. Various growth factors known in the art canbe combined with the SHIP inhibitors of the present invention in acombination therapy, e.g., to boost neutrophils/granulocytes, red bloodcells, and platelets in a subject. Further, the SHIP inhibitors of thepresent invention may be used to enhance blood stem cell harvest, e.g.,SHIP inhibitors have been shown to mobilize a radioprotective dose ofsuch stem cells from the bone marrow to the blood.

In view of the above, below are various other aspects of the presentinvention.

In another aspect, the present invention relates to a method of treatingmyelosuppression in a subject. This method includes the step ofadministering to the subject one or more SHIP1 inhibitor compound of thepresent invention under conditions effective to treat myelosuppressionin the subject.

In another aspect, the present invention relates to a method of treatinganemia in a subject. This method includes the step of administering tothe subject one or more SHIP1 inhibitor compound of the presentinvention under conditions effective to treat anemia in the subject.

In another aspect, the present invention relates to a method ofincreasing platelets in a subject. This method includes the step ofadministering to the subject one or more SHIP1 inhibitor compound of thepresent invention under conditions effective to increase platelets inthe subject.

In another aspect, the present invention relates to a method of aidingrecovery of a subject who has undergone a bone marrow transplant. Thismethod includes the step of administering to the subject one or moreSHIP1 inhibitor compound of the present invention under conditionseffective to increase production in the subject of blood cellcomponents, thereby aiding the post-bone marrow transplant recovery ofthe subject. This method can be used to aid the recovery of subjects whohave undergone an autologous bone marrow transplant or an allogenic bonemarrow transplant. In one embodiment, this method can further includeadministering at least one growth factor to the subject along with theone or more SHIP1 inhibitor compound.

In another aspect, the present invention relates to a method ofenhancing blood stem cell harvest from a subject. This method includesthe step of administering to the subject one or more SHIP1 inhibitorcompound of the present invention under conditions effective to mobilizestem cells in the subject from the bone marrow to the blood.

EXAMPLES

The following examples are intended to illustrate particular embodimentsof the present invention, but are by no means intended to limit thescope of the present invention.

Example 1 Synthesis of 3α-Acetamido-5α-Cholestane

The 3α-acetamido-5α-cholestane of the present invention can be madeusing the following synthetic scheme:

Example 2 Experimental Data Relating to 3α-Acetamido-5α-Cholestane

3α-Acetamido-5α-cholestane

The α-amine (0.29 g, 0.75 mmol) was dissolved THF (2.21 mL) in a roundbottom flask. Et₃N (0.12 mL, 0.90 mmol) was added dropwise and theresulting solution was cooled at 0° C. Acetyl chloride (0.06 mL, 0.83mmol) was added dropwise into the cooled solution which resulted on theformation of white precipitate. The milky white solution was stirredcontinuously for 15 min at 0° C. before allowing the reaction mixture towarm up to room temperature. THF (5 mL) was added and the dilutedsolution was washed with HCl (10 mL, 1 M), brine solution (10 mL), andH₂O (10 mL). The organic layer was collected, dried over Na₂SO₄, andconcentrated under reduced pressure. Recrystallization of the solidresidue using EtOH afforded amide (0.22 g, 65%) as off white solid.

IR (KBr): 3265, 2931, 2864, 2848, 1667, 1337 cm⁻¹. m.p.=215-216° C. ¹HNMR (300 MHz, CDCl₃): δ 5.71 (broad, 1H), 4.13 (broad, 1H), 1.99 (s,3H), 1.96 (t, J=3 Hz, 1H), 1.79 (m 1H), 1.60-1.65 (m, 2H), 1.45-1.60 (m,7H), 1.31-1.36 (m, 6H), 1.27-1.28 (m, 1H), 1.03-1.04 (m, 2H), 0.96-1.00(m, 5H), 0.94-0.96 (m, 1H), 0.87 (s, J=1.2 Hz, 3H), 0.85 (d, J=1.2 Hz,3H), 0.80 (s, 3H), 0.68-0.73 (m, 1H), 0.65 (s, 3H). ¹³C NMR (75 MHz,CDCl₃): δ 169.4, 56.7, 56.4, 54.7, 44.9, 42.7, 41.0, 40.2, 39.6, 36.3,36.1, 35.9, 35.5, 33.3, 33.0, 32.1, 28.6, 28.4, 28.1, 26.1, 24.3, 24.0,23.8, 23.0, 22.7, 20.9, 18.8, 12.2, 11.6

Example 3 Synthesis of the β-Amine Compound

The β-amine compound of the present invention can be made using thefollowing synthetic scheme:

Example 4 Analogs

Various analogs are contemplated as being SHIP inhibitors of the presentinvention, as described below:

Example 5 Synthetic Schemes

Below are various schemes relating to analogs contemplated as being SHIPinhibitors of the present invention, as described below:

Example 6 5α-Androstan-3β-ol

5α-Androstan-3β-ol

In a flame-dried flask, potassium hydroxide (1.58 g, 28.2 mmol) wasdissolved in ethylene glycol (10 mL) by heating. The solution was cooledat room temperature before adding trans-androsterone (2.00 g, 6.89 mmol)and hydrazine hydrate (0.98 mL, 20.2 mmol). The solution was heated toreflux at 208° C. After 23 h, the solution was cooled at roomtemperature before adding HCl (14.1 mL, 2M). It was extracted withCH₂Cl₂ (4×30 mL). The organic layer were collected, combined, dried overNa₂SO₄, and concentrated under reduced pressure. The resulting solidresidue was recrystallized in MeOH to afford 5α-androstan-3β-ol (1.56 g,82%). ¹H NMR (300 MHz, CDCl₃): d 3.58 (heptet, J=4.9 Hz, 1H), 1.76-1.82(m, 1H), 1.70-1.75 (m, 2H), 1.65-1.69 (m, 2H), 1.61-1.63 (m, 1H),1.57-1.60 (m, 1H), 1.52-1.57 (m, 2H), 1.47-1.50 (m, 1H), 1.40-1.45 (m,1H), 1.33-1.39 (m, 1H), 1.29-1.30 (m, 1H), 1.22-1.28 (m, 4H), 1.04-1.17(m, 4H), 0.9-1.02 (m, 1H), 0.85-0.93 (m, 2H), 0.80 (s, 3H), 0.68 (s, 3H)0.60-0.65 (m, 1H).

Example 7 3α-Azido-5α-Androstane

3α-Azido-5α-Androstane

In a 50 mL round bottom flask, 5α-androstan-3,3-ol (1.12 g, 4.05 mmol)was dissolved in THF (20 mL). PPh₃ (1.06 g, 4.04 mmol) was added intothe solution followed by DIAD (0.83 mL, 4.05 mmol). The resulting yellowsolution was stirred continuously at room temperature for 10 min beforeadding (PhO)₂PON₃ (0.88 mL, 4.05 mmol). The solution was stirredcontinuously at room temperature. After 24 h, the reaction mixture wasconcentrated and the residue was recrystallized to afford3α-azido-5α-androstane as a white solid (0.90 g, 74%). ¹H NMR (300 MHz,CDCl₃): δ 3.88 (p, J=2.8 Hz, 1H), 1.71-1.72 (m, 1H), 1.67-1.70 (m, 3H),1.59-1.64 (m, 2H), 1.57-1.53 (m, 3H), 1.45-1.52 (m, 3H), 1.36-1.42 (m,2H), 1.26-1.31 (m, 1H), 1.18-1.24 (m, 3H), 1.14-1.17 (m, 2H), 1.13-1.10(m, 1H), 0.85-1.03 (m, 2H), 0.79 (s, 3H), 0.72-0.77 (m, 1H), 0.69 (s,3H).

Example 8 3α-Amino-5α-Androstane

3α-Amino-5α-Androstane

In round bottom flask, LiAlH₄ (0.39 g, 9.83 mmol, 95%) was suspended inTHF (10 mL). The suspension was cooled at 0° C. using ice/H₂O bathbefore adding a solution of α-azide (0.90 g, 2.98 mmol) in THF (5 mL).The solution was warmed to room temperature and refluxed at 80° C. for 4h. The reaction was cooled to room temperature before diluting thesolution with THF (15 mL). The diluted reaction mixture was cooled at 0°C. and quenched using a Fieser method. The reaction mixture was stirredcontinuously until it turned into a milky white solution. The solutionwas then filtered through celite and washed with THF. The filtrate wasdried over Na₂SO₄ and concentrated under reduced pressure to afford3α-amino-5α-androstane (0.59 g, 72%). IR (KBr): 2926, 2855, 1472, 1378,1124, 753 cm⁻¹. ¹H NMR (300 MHz, CDCl₃): d 3.18 (broad, 1H), 1.71-1.73(m, 2H), 1.65-1.69 (m, 3H), 1.61-1.63 (m, 1H), 1.59-1.60 (m, 1H),1.55-1.57 (m, 2H), 1.50-1.53 (m, 1H), 1.40-1.45 (m, 3H), 1.30-1.32 (m,1H), 1.23-1.29 (m, 3H), 1.18-1.21 (m, 3H), 1.14-1.18 (m, 2H), 1.07-1.10(m, 2H), 0.89-1.99 (m, 2H), 0.78 (s, 3H), 0.69 (s, 3H).

Example 9 3α-Amino-5α-androstane hydrochloride

3α-Amino-5α-androstane hydrochloride

The α-amine 11 (0.20 g, 0.73 mmol) was dissolved in Et₂O (5 mL). Asolution of HCl in Et₂O (0.73 mL, 2 M) was added dropwise which resultedto the formation of precipitate. The solution was filtered and theprecipitate was collected, washed over Et₂O, and dried over vacuum toafford 3α-amino-5α-androstane hydrochloride (0.15 g, 65%) as a whitesolid. IR (KBr): 3320, 2945, 1619, 1495, 1443, 1379 cm⁻¹. ¹H NMR (300MHz, CDCl₃): d 8.45 (broad, 3H), 3.60 (broad, 1H), 1.84 (broad, 2H),1.62-1.69 (m, 8H), 1.51-1.58 (m, 4H), 1.37-1.44 (m, 1H), 1.23-1.29 (m,2H), 1.09-1.20 (m, 4H), 0.92-1.07 (m, 3H), 0.79 (s, 3H), 0.69 (s, 3H).

Example 10 3α-Acetamido-5α-Androstane

3α-Acetamido-5α-Androstane

The α-amine (0.20 g, 0.73 mmol) was dissolved THF (3 mL) in a roundbottom flask. Et₃N (0.12 mL, 0.88 mmol) was added dropwise and theresulting solution was cooled at 0° C. Acetyl chloride (0.05 mL, 0.80mmol) was added dropwise into the cooled solution which resulted on theformation of white precipitate. The milky white solution was stirredcontinuously for 15 min at 0° C. before allowing the reaction mixture towarm up to room temperature. THF (5 mL) was added and the dilutedsolution was washed with HCl (10 mL, 1 M), brine solution (10 mL), andH₂O (10 mL). The organic layer was collected, dried over Na₂SO₄, andconcentrated under reduced pressure. Recrystallization of the solidresidue using EtOH afforded 3α-acetamido-5α-androstane (0.05 g, 22%) aswhite solid. IR (KBr): 3264, 3077, 2933, 2834, 1637, 1558 cm⁻¹. ¹H NMR(300 MHz, CDCl₃): δ 5.70 (broad, 1H), 4.12 (m, 1H), 1.99 (s, 3H),1.72-1.76 (m, 1H), 1.68-1.71 (m, 2H), 1.62-1.66 (m, 2H), 1.60-1.62 (m,2H), 1.56-1.58 (m, 1H), 1.52-1.55 (m, 1H), 1.48-1.51 (m, 1H), 1.42-1.46(m, 1H), 1.36-1.39 (m, 1H), 1.29-1.34 (m, 2H), 1.23-1.27 (m, 1H), 1.21(d, J=3.0 Hz, 1H), 1.18-1.19 (m, 1H), 1.12-1.17 (m, 2H), 1.08-1.11 (m,1H), 1.00-1.06 (m, 1H), 0.92-0.97 (m, 1H), 0.84-0.90 (m, 1H), 0.81 (s,3H), 0.71-0.77 (m, 1H), 0.69 (s, 3H).

Example 11 3β-Tosyloxy-5α-Androstan-17-one

3β-Tosyloxy-5α-Androstan-17-one

In a 25 mL round bottom flask, trans-androsterone (1.00 g, 3.44 mmol)and p-toluenesulfonyl chloride (1.51 g, 7.91 mmol) was dissolved in inpyridine (4.30 mL). The reaction mixture was stirred continuously atroom temperature. After 24 h, the reaction mixture was quenched byadding H₂O (10 mL) and it was extracted with CH₂Cl₂ (3×20 mL). Allorganic layers were collected, combined together and washed over HCl(3×20 mL, 2 M), brine solution (3×20 mL), and H₂O (3×20 mL), dried overNa₂SO₄, and concentrated under reduced pressure afforded3β-tosyloxy-5α-androstan-17-one (1.33 g, 87%) as a white solid. ¹H NMR(300 MHz, CDCl₃): d 7.79 (dt, J=8.3, 1.9 Hz, 2H), 7.33 (dd, J=8.0, 0.5Hz, 2H), 4.42 (h, J=5.9 Hz, 1H), 2.44 (s, 3H), 2.38-2.47 (m, 1H),1.99-2.11 (m, 1H), 1.86-1.95 (m, 1H), 1.78-1.80 (m, 1H), 1.71-1.77 (m,2H), 1.65-1.69 (m, 1H), 1.56-1.64 (m, 3H), 1.44-1.55 (m, 3H), 1.30-1.31(m, 1H), 1.28-1.29 (m, 2H), 1.22-1.24 (m, 1H), 1.18-1.20 (m, 1H),1.04-1.16 (m, 1H), 0.85-1.00 (m, 2H), 0.84 (s, 3H), 0.80 (s, 1H),0.60-0.69 (m, 1H).

Example 12 3α-Azido-5α-Androstan-17-one

3α-Azido-5α-Androstan-17-one

A suspension of tosylate (1.33 g, 2.99 mmol) and NaN₃ (1.94 g, 29.9mmol) in DMSO (75 mL) was heated to reflux at 90° C. After approximately5 h, the reaction mixture was cooled at room temperature before addingH₂O (10 mL). The diluted solution was extracted with Et₂O (3×20 mL). Allorganic layers were collected, dried over MgSO₄, and concentrated underreduced pressure. The solid residue was recrystallized in EtOH to afford3α-azido-5α-androstan-17-one (0.28 g, 30%). ¹H NMR (300 MHz, CDCl₃): d3.88 (pentet, J=2.6 Hz, 1H), 2.43 (dd, J=10.3, 9.6 Hz, 1H), 2.00-2.12(m, 1H), 1.88-1.97 (m, 1H), 1.81-1.83 (m, 1H), 1.76-1.78 (m, 1H),1.66-1.72 (m, 2H), 1.62-1.65 (m, 1H), 1.51-1.56 (m, 2H), 1.39-1.49 (m,4H), 1.17-1.34 (m, 7H), 0.94-1.08 (m, 1H), 0.85 (s, 3H), 0.81 (s, 3H).

Example 13 3α-Amino-5α-androstan-17-one hydrochloride

3α-Amino-5α-androstan-17-one hydrochloride

In a flame dried flask, azide (0.28 g, 0.89 mmol) and PPh₃ (0.36 g, 1.37mmol) was dissolved in THF (15 mL). The solution was stirredcontinuously at room temperature for 18 h. H₂O (3 mL) was added and thesolution was heated to reflux at 80° C. After 1 h, the solution wascooled at room temperature. The organic layer was collected, dried overNa₂SO₄, and concentrated under reduced pressure. The residue wasdissolved Et₂O (7 mL) and a solution of HCl (0.89 mL, 2 M) was addedwhich resulted to formation of precipitate. The precipitate was filteredover filter paper, washed over Et₂O, and dried to afford3α-amino-5α-androstan-17-one hydrochloride (0.22 g, 76%) as white solid.IR (KBr): 3326, 2923, 1737, 1496, 1455, 731 cm⁻¹. ¹H NMR (300 MHz,CDCl₃): δ 8.42 (broad, 3H), 3.61 (broad, 1H), 2.42 (dd, J=11.1, 8.7 Hz,1H), 2.00-2.13 (m, 1H), 1.86-1.94 (m, 2H), 1.76-1.83 (m, 3H), 1.44-1.64(m, 7H), 1.19-1.38 (m, 6H), 0.95-1.13 (m, 2H), 0.84 (s, 3H), 0.81 (s,3H).

Example 14 3α-Azidocholest-5-ene

3α-Azidocholest-5-ene

Cholesterol (7.76 mmol, 3.0 g) and triphenylphosphine (7.76 mmol, 2.04g) were dissolved in 77.6 mL of anhydrous tetrahydrofuran. Diisopropylazodicarboxylate (7.76 mmol, 1.5 mL) was then added dropwise. Afterstirring the orange mixture for a few minutes, diphenylphosphoryl azide(7.76 mmol, 1.68 mL) was added dropwise. After 24 hours, the pale yellowreaction mixture was concentrated. Purification by silica gelchromatography (100% hexanes) afforded 3α-azidocholest-5-ene (2.14 g,67%) as a white solid. mp 110-112° C.; TLC R_(f)=0.87 (20% ethylacetate/hexanes); IR (thin film) 2946, 2914, 2845, 2083 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ 5.42-5.40 (m, 1H), 3.89 (t, 1H, J=2.9 Hz), 2.58-2.49(m, 1H), 2.23-2.16 (m, 1H), 2.16-1.93 (m, 2H), 1.89-1.05 (m, 24H), 1.02(s, 3H), 0.93 (d, 3H, J=6.5 Hz), 0.88 (d, 6H, J=6.6 Hz), 0.69 (s, 3H).

Example 15 3α-Aminocholest-5-ene

3α-Aminocholest-5-ene

3α-Azidocholest-5-ene (4.62 mmol, 1.9 g) was dissolved in 154 mL ofanhydrous diethyl ether. Lithium aluminum hydride (46.2 mmol, 1.75 g)was then added in one portion. After 30 hours, the reaction mixture wascooled to 0° C. 1.75 mL of deionized water was then added dropwise.After stirring for five minutes, 1.75 mL of 15% aqueous NaOH was addeddropwise. After stirring for another five minutes, 5.25 mL of deionizedwater was added dropwise. The reaction was then stirred until all thesalts turned white. Immediately afterwards, the reaction was filtered,dried (Na₂SO₄), and concentrated to afford 3α-Aminocholest-5-ene (1.57g, 93%) as a white solid. mp 104-106° C.; IR (thin film) 3367, 3343,2931, 1557 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) d 5.37-5.34 (m, 1H), 3.15 (t,1H, J=3.2 Hz), 2.61-2.54 (m, 1H), 2.04-1.74 (m, 6H), 1.63-1.03 (m, 21H),1.00 (s, 3H), 0.91 (d, 3H, J=6.5 Hz), 0.86 (d, 6H, J=6.6 Hz), 0.67 (s,3H).

Example 16 3α-Aminocholest-5-ene Hydrochloride

3α-Aminocholest-5-ene Hydrochloride

3α-Aminocholest-5-ene (1.61 mmol, 0.59 g) was dissolved in 2 mL ofanhydrous diethyl ether. Hydrogen chloride (2.0 M in diethyl ether)(3.22 mmol, 1.61 mL) was then added. After 3 hours, a white precipitateformed. The reaction was then filtered and the solid was washed withdiethyl ether to afford 3α-aminocholest-5-ene hydrochloride (0.31 g,46%) as a white solid. mp 293-295° C.; IR (thin film) 2947 cm⁻¹; ¹H NMR(300 MHz, CDCl₃) d 8.25 (s, 3H), 5.52 (d, 1H, J=4.3 Hz), 3.58 (s, 1H),2.61 (d, 1H, J=14.6 Hz), 2.36 (d, 1H, J=15.0 Hz), 2.02-1.07 (m, 26H),1.01 (s, 3H), 0.91 (d, 3H, J=6.3 Hz), 0.86 (d, 6H, J=6.6 Hz), 0.67 (s,3H).

Example 17 3α-Aminocholest-5-ene Citrate

3α-Aminocholest-5-ene Citrate

3α-Aminocholest-5-ene (0.82 mmol, 300 mg) was dissolved in 1.64 ml oftetrahydrofuran. Citric acid (0.82 mmol, 158 mg) was dissolved in 0.82ml of tetrahydrofuran. The solution of citric acid was added dropwise tothe solution of cholesterol amine. The mixture was stirred until thesolution became very cloudy (approximately 15 minutes). The solution wasvacuum filtered. The resulting white solid was washed withtetrahydrofuran, collected, and dried under high vacuum for 12 hours toproduce 298 mg of the 3α-aminocholest-5-ene citrate in 63% yield. mp:172-174° C.; IR (thin film): 3469, 2954, 2247, 1714, 1591 cm⁻¹; ¹H NMR(300 MHz, CD₃OD) δ: 5.53 (d, 1H, J=5.2 Hz), 3.55 (s, 1H), 2.84-2.70 (m,4H), 2.80-2.70 (m, 1H), 2.19-1.0 (m, 28H), 1.07 (s, 3H), 0.95 (3H, J=6.5Hz), 0.89 (d, 6H, J=6.6 Hz), 0.73 (s, 3H).

Example 18 Synthesis and Identification of 3AC Derivatives withIncreased Solubility and Potency

The solubility of 3AC was assessed by calculating the distributioncoefficient (CLogD). This calculation estimates the CLogD for 3AC at7.17 indicating the molecule is very lipophilic. Thus, we have begun todevelop novel 3AC analogs with increased aqueous solubility. One ofthese compounds, 3A5AS, has a CLogD of 3.33. Lipinski's rules (a commonmeasure of small molecule pharmacokinetics) recommend a ClogD of <5 forin vivo applications. The chemical modifications made to derive 3A5AShave not altered its ability to inhibit SHIP1 as it retains equalinhibitory activity in vitro (FIG. 6A) and, surprisingly, it is morepotent when used on intact cells, as it is substantially more cytotoxicfor leukemia cells (FIG. 6B). 3A5AS is also more potent at inducing MIRcell numbers in vivo as it can induce a comparable MIR cell increase at1504 as opposed to 60 μM 3AC (FIG. 6C,D).

REFERENCES CITED

Citation of a reference herein shall not be construed as an admissionthat such reference is prior art to the present invention. Allreferences cited herein are hereby incorporated by reference in theirentirety. Below is a listing of references cited herein with referencenumber indicators:

-   1. Wang J W, et al. (2002) Influence of SHIP on the NK repertoire    and allogeneic bone marrow transplantation. (Translated from eng)    Science 295(5562):2094-2097 (in eng).-   2. Ghansah T, et al. (2004) Expansion of myeloid suppressor cells in    SHIP-deficient mice represses allogeneic T cell responses.    (Translated from eng) J Immunol 173(12):7324-7330 (in eng).-   3. Wahle J A, et al. (2006) Cutting edge: dominance by an    MHC-independent inhibitory receptor compromises NK killing of    complex targets. (Translated from eng) J Immunol 176(12):7165-7169    (in eng).-   4. Paraiso K H, Ghansah T, Costello A, Engelman R W, & Kerr W    G (2007) Induced SHIP deficiency expands myeloid regulatory cells    and abrogates graft-versus-host disease. (Translated from eng) J    Immunol 178(5):2893-2900 (in eng).-   5. Kerr W G (2008) A role for SHIP in stem cell biology and    transplantation. Curr Stem Cell Res Ther 3(2):99-106.-   6. Helgason C D, et al. (1998) Targeted disruption of SHIP leads to    hemopoietic perturbations, lung pathology, and a shortened life    span. Genes & Development 12(11):1610-1620.-   7. Rauh M J, et al. (2005) SHIP represses the generation of    alternatively activated macrophages. Immunity 23(4):361-374.-   8. Takeshita S, et al. (2002) SHIP-deficient mice are severely    osteoporotic due to increased numbers of hyper-resorptive    osteoclasts. Nat Med 8(9):943-949.-   9. Franke T F, Kaplan D R, Cantley L C, & Toker A (1997) Direct    regulation of the Akt proto-oncogene product by    phosphatidylinositol-3,4-bisphosphate [see comments]. Science    275(5300):665-668.-   10. Jain S K, et al. (1996) PI 3-kinase activation in    BCR/abl-transformed hematopoietic cells does not require interaction    of p85 SH2 domains with p210 BCR/abl. (Translated from eng) Blood    88(5):1542-1550 (in eng).-   11. Ivetac I, et al. (2009) Regulation of PI(3)K/Akt signalling and    cellular transformation by inositol polyphosphate 4-phosphatase-1.    (Translated from eng) EMBO Rep 10(5):487-493 (in eng).

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A SHIP inhibitor compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein formula (I) is as follows:

wherein R¹ is a straight chain C₁-C₄ alkyl or C₁-C₄ haloalkyl; R² is hydrogen, methyl, or halomethyl; R³ is hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, C₁-C₄ haloalkyl, or C₁-C₄ alkenyl; R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, C₁-C₄ alkyl, or benzyl; R⁵ represents a divalent oxo atom, or two hydrogen atoms, or one hydrogen atom together with an alkyl group; X¹ is selected from the group consisting of hydrogen, hydroxy, mercapto, alkoxy, aryloxy, alkylthio, arylthio, alkylcarbonamido, alkoxycarbonamido, arylcarbonamido, aryloxycarbonamido, alkylsulfonamido, arylsulfonamido, substituted or unsubstituted amino, and aminoalkyl; and each X² individually represents a divalent oxo atom or two hydrogen atoms; with the proviso that X¹ cannot be a primary amino group when: R¹ and R² are each methyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents one hydrogen atom together with a 1,5-dimethylhexyl alkyl group. 2-15. (canceled)
 16. A pharmaceutical composition comprising: a compound according to claim 1; and a pharmaceutically acceptable carrier. 17-33. (canceled)
 34. A method of inhibiting SHIP activity in a cell, said method comprising: contacting the cell expressing SHIP1 or SHIP2 with a SHIP inhibitor compound of formula (I), or a pharmaceutically acceptable salt thereof, wherein formula (I) is as follows:

wherein R¹ is a straight chain C₁-C₄ alkyl or C₁-C₄ haloalkyl; R² is hydrogen, methyl, or halomethyl; R³ is hydrogen, substituted or unsubstituted amino, C₁-C₄ alkyl, C₁-C₄ haloalkyl, or C₁-C₄ alkenyl; R⁴ is hydrogen, hydroxy, substituted or unsubstituted amino, C₁-C₄ alkyl, or benzyl; R⁵ represents a divalent oxo atom, or two hydrogen atoms, or one hydrogen atom together with an alkyl group; X¹ is selected from the group consisting of hydrogen, hydroxy, mercapto, alkoxy, aryloxy, alkylthio, arylthio, alkylcarbonamido, alkoxycarbonamido, arylcarbonamido, aryloxycarbonamido, alkylsulfonamido, arylsulfonamido, substituted or unsubstituted amino, and aminoalkyl; and each X² individually represents a divalent oxo atom or two hydrogen atoms; with the proviso that X¹ cannot be a primary amino group when: R¹ and R² are each methyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents one hydrogen atom together with a 1,5-dimethylhexyl alkyl group.
 35. A method according to claim 34, wherein R¹ and R² are each methyl, and R³ and R⁴ are each hydrogen.
 36. A method according to claim 34, wherein R⁵ represents one hydrogen atom together with a 1,5-dimethylhexyl group.
 37. A method according to claim 34, wherein R³ and R¹³ are each hydrogen.
 38. A method according to claim 34, wherein X¹ is selected from the group consisting of hydroxy, mercapto, alkoxy, aryloxy, alkylthio, and arylthio.
 39. A method according to claim 34, wherein X¹ is selected from the group consisting of alkylcarbonamido, alkoxycarbonamido, arylcarbonamido, aryloxycarbonamido, aminocarbonamido, and hydrazinocarbonamido.
 40. A method according to claim 39, wherein X¹ is acetamido.
 41. A method according to claim 34, wherein X¹ is selected from the group consisting of alkylsulfonamido, arylsulfonamido, aminosulfonamido, and hydrazinosulfonamido.
 42. A method according to claim 34, wherein X¹ is selected from the group consisting of (C₁-C₄ alkyl)carbonyloxy, (C₁-C₄ alkoxy)carbonyloxy, arylcarbonyloxy, aryloxycarbonyloxy, and aminocarbonyloxy.
 43. A method according to claim 34, wherein X¹ is a secondary or tertiary amino group that includes at least one C₁-C₄ alkyl, C₅-C₆ cycloalkyl, aryl, or heterocyclic substituent, or combinations thereof.
 44. A method according to claim 34, wherein the secondary or tertiary amino group includes at least one optionally substituted C₁-C₄ alkyl moiety.
 45. A method according to claim 34, wherein X¹ is an aminoalkyl group, wherein amino is an unsubstituted or a substituted secondary or tertiary amino, and n is an integer from 1 to
 4. 46. A method according to claim 34, wherein X¹ is a divalent oxygen moiety, ═O, or a divalent N-hydroxyamino moiety, ═NOH.
 47. A method according to claim 34, wherein X¹ is an amino group, except when: R¹ and R² are each methyl; X², R³, R⁴, and R¹³ are each hydrogen; and R⁵ represents one hydrogen atom together with an alkyl group, where the alkyl group is 1,5-dimethylhexyl.
 48. A method according to claim 34, wherein said compound of formula (I) is a compound of a formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof, wherein X═NR₂, NRCOR, NHCONR₂, OR, SR, OCOR, OCONR₂, or NHCNHNH₂, and wherein R═H, alkyl, cycloalkyl, aryl, or benzyl.
 49. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 treats or prevents graft versus host disease in a transplant recipient.
 50. The method of claim 49, wherein contacting the cell expressing SHIP1 or SHIP2 with a SHIP inhibitor compound comprises administering to the transplant recipient the SHIP inhibitor compound in a pharmaceutically effective amount after the transplantation.
 51. The method of claim 49, wherein contacting the cell expressing SHIP1 or SHIP2 with a SHIP inhibitor compound comprises: isolating blood derived cells, bone marrow transplants, or organ transplants; and contacting the isolated blood derived cells, bone marrow transplants, or organ transplants with the SHIP inhibitor compound, whereby the treatment of transplants inactivates T-lymphocytes therein.
 52. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 inhibits tumor growth and metastasis.
 53. The method of claim 52, wherein the cell is a human breast cancer cell.
 54. The method of claim 53, wherein contacting the cell expressing SHIP1 or SHIP2 with a SHIP inhibitor compound comprises administering to a subject having breast cancer the SHIP inhibitor compound.
 55. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 treats a hematologic malignancy in a subject.
 56. The method of claim 55, wherein the hematologic malignancy is multiple myeloma, and treating the hematologic malignancy comprises inducing apoptosis of multiple myeloma cells by contacting the multiple myeloma cells with the SHIP inhibitor compound.
 57. The method of claim 56, wherein contacting the multiple myeloma cells with the SHIP inhibitor compound comprises administering to the subject the SHIP inhibitor compound.
 58. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 boosts production of all key blood cell types, white blood cells or lymphocytes.
 59. The method of claim 58, wherein a subject having anemia is treated by boosting the production of red blood cells in the subject by administering to the subject the SHIP inhibitor compound under conditions effective to treat anemia in the subject.
 60. The method of claim 58, wherein the production of platelets in a subject is boosted by administering to the subject the SHIP inhibitor compound under conditions effective to increase platelets in the subject.
 61. The method of claim 58, wherein a subject having myelosuppression is treated by administering to the subject the SHIP inhibitor compound under conditions effective to treat myelosuppression in the subject.
 62. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 aids recovery of a subject who has undergone a bone marrow transplant, said inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 comprising: administering to the subject the SHIP inhibitor compound under conditions effective to increase production in the subject of blood cell components, thereby aiding the post-bone marrow transplant recovery of the subject, wherein the bone marrow transplant is selected from the group consisting of an autologous bone marrow transplant and an allogenic bone marrow transplant.
 63. The method of claim 34, wherein inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 enhances blood stem cell harvest from a subject, said inhibiting SHIP activity in a cell expressing SHIP1 or SHIP2 comprising: administering to the subject one or more SHIP1 inhibitor compound according to claim 1 under conditions effective to mobilize stem cells in the subject from the bone marrow to the blood.
 64. The method of claim 34, wherein the SHIP inhibitor compound is provided in an amount effective to inhibit SHIP1 but not to inhibit SHIP2 or PTEN. 